1. Introduction

Ionic liquids (ILs) are the salts of organic cations in the liquid state. The most common cations forming ionic liquids are pyridinium, piperidinium, and imidazolium. In addition, ammonium, guanidinium, sulfonium, phosphonium, or oxonium having various alkyl chains can also form ionic liquids. The counteranions can be small organic or inorganic ions, such as halogen-based anions (Cl⁻, Br⁻, BF₄⁻, and AlCl₄⁻), bis(trifluoromethanesulfonyl)imide, [(CF₃SO₂)₂N]⁻, trifluoroacetate [CF₃COO⁻], or trifluoromethanesulfonate [CF₃SO₃⁻] [1]. Examples of some common organic cations and anions in the composition of ionic liquids are given in Figure 1.

Figure 1.
Chemical structures, abbreviations, and names of several common ionic liquids.

ILs are currently used as an alternative to the organic solvents for various analytical processes [1], considering that they can be more environmentally friendly solvents than the classic organic solvents, due to their low volatility, flammability, and toxicity. The liquid property of these ionic compounds can be explained by the role of the short-range interactions and ion packing, combined with their long molecular structure [2]. Owing to the ability of ionic liquids to solvate the compounds of widely varying polarity [3], they are more and more utilized in separation processes. Examples of analytical separations based on ILs are liquid-liquid extraction (single drop microextraction, dispersive liquid-liquid microextraction, and hollow fiber membrane liquid phase) [4, 5, 6], solid-phase extraction and microextraction [7], gas chromatography (stationary phases based on ILs) [8, 9], electrophoresis [10, 11], and high-performance liquid chromatography (HPLC), discussed further in this chapter.

The properties of ILs in the separation process depend on their interaction properties with surrounding molecules or surfaces, ion exchanging, H-bonding, ion-dipole, dipole-dipole, π-π interactions, hydrophobic, and hydrophilic interactions [12]. The interaction properties of ILs depend on the nature of both their anion and cation. The H-bonding interactions with water, by means of anion, determine the hydrophobic or hydrophilic character of IL. For example, for the IL based on the cation 1-butyl-3-methylimidazolium [C4mim], its hydrophobicity depends on the anion size: for anions, such as NO₃⁻, or BF₄⁻, the interactions with water molecules are strong, and consequently, the IL becomes hydrophilic, while for anions PF₆⁻ or bis(trifluoromethanesulfonyl)-imide [(CF₃-SO₂)₂N⁻ or NTf2], the IL assembly becomes hydrophobic [13]. The nonpolar (hydrophobic) parts of ILs interact preferentially with nonpolar compounds by van der Waals forces, while the interface between the polar and nonpolar parts of ILs can interact by dipole-dipole with polar compounds, such as halogenated hydrocarbons or acetone [14]. Besides solubility in different solvents, the transport properties (viscosity, diffusion, and conductivity) can be described using theories on the structure–property correlations [15].

2. Stationary phases with attached structures of ionic liquids

There are a large variety of stationary phases containing ILs as an active functional group. Owing to their complex structure, these stationary phases are used in different separation mechanisms, including reversed-phase (RP), normal-phase (NP), hydrophilic interaction liquid chromatography (HILIC), ion-exchange chromatography (IEC), or combination of these mechanisms, known as mixed-mode separations. Generally, the stationary phases in HPLC having in their structure moieties of ionic liquids are synthetized having silica gel (type B) or derivatized silica gel as starting materials. Stationary phase can also be obtained by the polymerization of ionic liquids or by monolithic technologies. The most common types of IL-based stationary phases are bonded phases with a single-cation ionic liquid, obtained as spherical particles [16]. Ionic liquid stationary phases with multication ionic liquids and mixed functionalities between ionic liquids and hydrophobic or polar groups are also known in liquid chromatography [16].

The simplest method to introduce an IL moiety to the silica support is based on the two-step synthesis route illustrated in Figure 2. The first reaction is generally used to obtain chloropropyl silica, as intermediate material, from silica gel and 3-chloropropyltrimethoxysilane. The second reaction, between substituted imidazole or pyridine and chloropropyl silica gel, takes place in toluene under reflux [17, 18]. Longer linker than propyl is also used in attaching imidazole to the silica surface, such as octyl [19]. Imidazolium-based ILs with various functionalities are produced in order to obtain stationary phases with different retention properties: methyl, benzyl, and butylsulphonic [19].

Figure 2.
The synthesis route for attaching imidazolium and pyridinium IL structure to the surface of silica gel (R can be C8, C10, C18, benzyl, or antranyl).

Direct reaction between silica gel and imidazole-based derivatization reagent is also possible. Thus, the reaction between 3-bromopropyl-trimethoxysilane and 1-alkylimidazole can be used to attach IL to the silica gel surface [20, 21], as schematically shown in Figure 3.

Figure 3.
Reaction between a functionalized imidazolium salt with silanol group from silica gel surface (adapted from [21]).

Thiopropyl silica gel can also be used for obtaining a stationary phase with a single-cation IL based on a synthesis similarly to the schema presented in Figure 2, but based on addition reaction of thio group to the allyl radical of imidazolium-based IL (known also as “thiol-ene” click reaction). The structural feature of this stationary phase [22], used in the HILIC separation mechanism, is given in Figure 4.

Figure 4.
Stationary phase synthetized from thiopropyl silica gel (obtained from silica gel in the first reaction) by attaching an imidazolium-based IL (adapted from [23]).

Monolithic stationary phases are also reported in the literature [24], also known as supported poly(ionic liquid)s (SPILs) [25]. The most convenient method to prepare these stationary phases is based on monolithic support obtained from glycidyl methacrylate as monomer, using ethylene glycol dimethacrylate as crosslinker [26, 27]. Usually, the polymerization reaction is thermally initiated 2,2′-azobis-isobutyronitrile) by ultraviolet (UV) or γ radiation in a porogenic solvent (generally, they are the mixtures of cyclohexanol and dodecanol) [28]. The crosslinked structure contains epoxy groups, which are very reactive and can be used to react with imidazolium-based compound leading to more complex structures containing IL moiety at its surface [29]. An example of obtaining such monoliths is given in Figure 5.

Figure 5.
Synthesis route to obtain a monolithic stationary phase based on glycidyl methacrylate and imidazolium moiety on its surface.

Another possibility of obtaining polymers with ILs grafted to its surface is described in Figure 6. In this procedure, the monomer contains the imidazolium IL, which is then grafted to the surface of silica gel having thiopropyl as linker [30].

Figure 6.
Polymeric structure containing imidazolium IL grafted to the surface of thiopropyl silica surface (adapted from [30]).

Based on this route, two types of stationary phases were obtained bearing two different anions: 1-[2-alkyl(mercaptopropyl)]-3-octadecyl-imidazolium bromide and 1-[2-alkyl(mercaptopropyl)]-3-octadecyl-imidazolium p-dimethylaminoazobenzenesulfonate [31]. Their chemical structures are shown in Figure 7.

Figure 7.
Structure of 1-[2-alkyl(mercaptopropyl)]-3-octadecyl-imidazolium bonded to silica gel obtained by polymerization.

The copolymerization of anionic (vinylsulphonic acid) and cationic (vinylimidazolium substituted with long alkyl chain) monomers that were grafted on thiopropyl silica gel was used to obtain the stationary phase with mixed-mode characters, hydrophilic due to the IL moiety and reversed-phase due to the long alkyl chain attached to the imidazolium ring [32]. A structure of such material, where the radical R is typically a long alkyl chain (C18), is shown in Figure 8. These materials exhibited an increased stability and improved peak shape of separated compounds, such as polycyclic aromatic hydrocarbons, bases, and flavonoids.

Figure 8.
Chemical structure of copolymerized vinylsulphonic acid and R-substituted vinylimidazolium grafted to the mercaptopropyl silica gel (adapted from [32]).

A novel facile and efficient immobilization strategy was introduced on the basis of the reaction between an isocyanate derivative of IL and aminopropyl silica gel as mesoporous silica spheres. The result of this reaction is a multifunctional silica material used as HPLC stationary phase in the mixed-mode mechanism, with urea polar functionality and imidazolium salt bearing aliphatic chains of different lengths or aromatic groups [33]. The main derivatization reaction of aminopropyl silica gel with an isocyanate reagent bearing imidazolium salt is presented in Figure 9.

Figure 9.
Reaction between aminopropyl silica gel and isocyanate generating IL structure on silica gel surface (adapted from [33]).

There have been synthetized stationary phases containing three ILs attached to mercaptopropyl silica gel with different anions [34]. Two examples of stationary phases used in reversed-phase and hydrophilic separations are presented in Figure 10.

Figure 10.
Structure of tricationic ionic liquid attached to silica gel as HPLC stationary phases.

More complex structures containing IL moieties and used as stationary phases in HPLC are reported by the literature [16, 21]. An example of such a complex stationary phase is based on the use of graphene oxide, which plays the role of linker between ILs and the silica support, which is easy to make further chemical modification [35]. Besides silica gel, or organic monoliths used as stationary phase supports, there are attempts to use zirconia as support to improve their thermal stability [36]. Stationary phases with ILs with chiral properties are synthetized from cyclodextrin bonded on silica gel, where the positively charged imidazole structure provides electrostatic interactions with opposite charged groups from analytes. Thus, chiral ILs functionalized β-cyclodextrins (β-CDs) were synthetized by treating 6-tosyl-β-cyclodextrin with 1,2-dimethylimidazole, which were bonded to silica gel to obtain chiral stationary phases (CSPs) to be used in high-performance liquid chromatography. There are applications when they have been used in separating different chiral aromatic alcohol derivatives and racemic drugs in mobile phase based on acetonitrile [37].

Stationary phases based on other IL moieties have been recently reported by the literature [38]. For example, phosphonium-based ionic stationary phase used in the mixed-mode mechanism was synthetized by polymerization from trioctyl(allyl)phosphonium bromide (denoted by [P888Allyl]Br, obtained at its turn from trioctylphosphine and allyl bromide) and mercaptopropyl silica gel microsphere, based on the principle of thiol-ene click reaction, described schematically in Figure 4. This stationary phase proved useful in separating the mixtures of uracil, uridine, adenosine, cytidine, and cytosine, various drugs, and mixtures of hydrophobic compounds (e.g., series from benzene to butylbenzene).

3. Ionic liquids as additives in mobile phase

The addition of ionic liquids in mobile phase increases its polarity, and this parameter can be useful in some separation mechanisms. On the other hand, the presence of these species in mobile phase can negatively influence the spectral domain used for UV-Vis detection. One of the main applications of these additives is to suppress the interference of residual silanols in the retention mechanism based on hydrophobic stationary phases.

The influence of residual silanols from the surface of reversed-phase stationary phases, based on derivatized silica on the retention of basic compounds is known to produce peak asymmetry and tailing [39]. A solution to improve the shape of peaks and eliminate peak tailing is the addition of ionic liquids in the composition of mobile phase, which have silanol suppressing properties [40]. This is based on the competition between the two adsorption equilibria, of ionic liquid cation and of basic analytes to the silanols, and thus, the basic analytes are less retained on these sites from the stationary phase surface. In practice, the concentration of ionic liquids is higher than that of analyte, and the analyte is practically excluded from the interaction with silanols. This effect has been observed in different applications, such as, for example, the separation of catecholoamines [41], β-lactam antibiotics [42], nucleotides [43], ephedrine derivatives [44], and fluoroquinolone [45], using imidazolium or pyridinium tetrafluoroborate ILs. However, the HPLC retention behavior of analytes in the presence of ILs in the mobile phase is influenced by both the cation and the anion due to their dissociation in aqueous medium [46].

The effect of ILs as additive on the retention of analytes depends on their polarity. The cation of IL can form ion pairs with the analytes as anions, and consequently, their retention can be increased in reversed-phase HPLC. On the other hand, the retention of organic cations is decreased by the presence of IL cation. For a complex mixture, the retention order and separation selectivity can be modified by the presence of IL in the composition of mobile phase. One example is given in Figure 11, where the separation of four compounds from a pharmaceutical formulation by the ion-pairing mechanism with sodium hexane sulphonate is carried out in the presence and absence of 1-butyl-1-methyl-pyrrolidinium tetrafluoroborate [47]. Usually, the increase of IL concentration in the mobile phase leads to an increase of analyte retention [48].

Figure 11.
Comparison between the retention of four pharmaceutical compounds (metamizole Na, metamizole impurity, fenpiverine bromide, and pitofenone hydrochloride, in the elution order), when the aqueous mobile phase contains: (1) both sodium hexane sulphonate and ionic liquid at 10 mM and (2) only 10 mM sodium hexane sulphonate (pH of the mobile phase is 3).

4. Retention behavior of ionic liquids studied as analytes in HPLC mechanisms

Separation between ionic liquids as analytes or between them and other organic species is possible by HPLC due to the differentiation in interacting with the stationary phase. The injection of samples containing ionic liquids as analytes in organic solvents has been studied by several chromatographic mechanism discussed as follows. The main possible interactions of ionic liquids, exemplified for imidazolium-based ILs with various moieties from HPLC stationary phases leading to retention, are illustrated in Figure 12. These interactions depend on the type of the stationary phase and, in many cases, on the composition of the mobile phase [49].

Figure 12.
Common intermolecular interactions between IL cation and silica gel (electrostatic interactions), C18 silica gel (van der Waals forces), and phenyl silica gel stationary phases (by cation-π interactions).

The retention on the octadecyl silica surface in the absence of ion pairing agents has revealed strong interactions between imidazolium-based cations and stationary phase, which can be used in their separation from mixtures. For a series of alkyl-imidazolium ionic liquids, the dependence of the retention factor (k), expressed as logarithm, and the number of carbon atoms from alkyl chain (n_C) is described as follows:

logk=αnC+βE1

where the parameters α and β are dependent on the nature of the anion part of the ionic liquids [50]. The dependence of the retention factor of a certain ionic liquid on the mobile phase composition is described as follows:

logk=a+bCo.m.E2

where C_o.m. represents the concentration of the organic modifier from the mobile phase (as percentage), and a and b are regression parameters calculated from the linear representation between the logarithm of the experimental retention factor and different mobile phase compositions used for the retention study. According to Eq. (2), the retention of IL species can be enhanced by decreasing the organic modifier (acetonitrile, methanol, and i-propanol) content in the mobile phase.

Another possibility to separate ionic liquids is the utilization of the ion-pairing mechanism. Owing to their positive charge, these ionic species can form ion pairs with opposite charged species, such as, for example, the alkylsulphonate anions, which can be interact by van der Waals forces with hydrophobic stationary phases (C8, C18, or even pentafluorophenylpropyl silica-based stationary phase) [51].

There are two models explaining the retention of ILs on hydrophobic stationary phases by the ion-pairing mechanism. The partition model supposes the formation of ion pair between IL+ and alkylsulphonate anion in the bulk of mobile phase, followed by the partition of the ion pair between the two phases, as represented by the following equilibria:

IL⁺ + CH₃-(CH₂)_n-SO₃⁻ ⇆ IL⁺⁻O₃S-(CH₂)_n-CH₃.

(IL⁺⁻O₃S-(CH₂)_n-CH₃)_mo.ph. ⇆ (IL⁺⁻O₃S-(CH₂)_n-CH₃)_st.ph..

The adsorption model [52] considers that the alkylsulphonate anion is adsorbed on the stationary phase surface due to its hydrophobic chain, and the resulted charged stationary phase can interact electrostatically with the IL cation, according to the following simple equilibria:

[CH₃-(CH₂)_n-SO₃⁻]_mo.ph. ⇆ [CH₃-(CH₂)_n-SO₃⁻]_st.ph.

(IL⁺)_mo.ph. + [CH₃-(CH₂)_n-SO₃⁻]_st.ph. ⇆ [CH₃-(CH₂)_n-SO₃⁻IL⁺]_st.ph..

In many cases, the graphical representations between the base 10 logarithm of the retention factor (log k) for the ionic liquid and the volume percentage of methanol in mobile phase (C_o.m.) showed a characteristic “U” shape with a minimum value within the studied interval of mobile phase composition. This experimental shape can be mathematically described by a second-order polynomial equation between log k and C_o.m., as follows:

logk=a+bCo.m.+cCo.m.2E3

where a is the intercept representing the extrapolated value of the retention factor (k_w), which corresponds to a mobile phase composed of only the aqueous component (a = log k_w for C_o.m. = 0), while b and c can be calculated from the polynomial regression applied to this functional dependence. Mathematically, this dependence has a minimum point characterized by the value of C_o.m., denoted by C^min, and the value of the retention factor, that is, min(log k), according to following equations:

Cmin=−b2candminlogk=a−b24cE4

This minimum corresponds to the mobile phase composition that allows the lowest affinity of the ion pairs between studied ionic liquids and alkylsulphonate for the octadecyl silica surface. Some experimental curves for the retention behavior of two ILs by ion pairing with C6, C7, and C8 alkylsulphonate anions [53] are illustrated in Figure 13.

Figure 13.
Comparative dependences for log k versus C_o.m., for the three alkylsulphonates as ion pairing agents, depicted for 1-butylpiridynium (BuPy⁺) bromide and 1-allyl-3-methylimidazolium (All3MeIm⁺) chloride [53].

Ion chromatography is another HPLC technique used for the analysis of ionic liquids [54]. Depending on which part of ILs is analyzed, the selection of the stationary phase is done to separate anions or cations. For example, the mixtures of ionic liquids with tetrafluoroborate [BF₄]⁻, hexafluorophosphate [PF₆]⁻, and bis(trifluoromethylsulfonyl)imide (triflimide) [NTf₂]⁻ anions combined with several cations based on imidazole, pyridine, and tetrahydrothiophene could be analyzed for the anion purity without any influence of IL cation, by means of anion chromatography, using as eluent Na₂CO₃/NaHCO₃ in water-acetonitrile solutions [55]. The separation of IL cation can be performed by stationary phases with strong cationic exchanging properties, but the interaction between IL cations and stationary phase is rather complex, depending on the composition of mobile phase. The elution of mixtures of IL cations at different mobile phase compositions revealed a separation mechanism based on cation exchange, combined with nonspecific hydrophobic interactions [56].

5. Perspectives in utilization of ILs as materials for HPLC

Ionic liquids are more and more of interest in separation science. Analytical methods in high-performance liquid chromatography make use of these compounds as stationary phases having them on their surface as distinct functionalities For this purpose, more innovative stationary phases are synthetized by various approaches, involved in almost all the separation mechanisms in HPLC. Owing to the more complex structure of the IL-based stationary phases, the separation mechanism is more complex than with conventional stationary phases for reversed-phase, hydrophilic, chiral, or ionic HPLC. Ionic liquids are known as additive components in the mobile phase, and in many situations, they have improvements on the separation performances by the reduction of silanophilic interactions in liquid chromatography [57]. Owing to their charge, these additives may play the role of ion-pairing reagent and enhance the retention of dissociable compounds in reversed-phase HPLC. The interest in this class of compounds can be seen from the more increasing number of publications (research articles, reviews, and book chapters) in the last decade. This is why their perspective in liquid chromatography is not only a hope [58] but also a necessity for solving different complex problems with already proved advantages. Combinations with other materials may lead to more promising materials useful in separation applications [59].

Marc ABRAMOWICZ

Chapters authored

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